Diagnostic preparation for the detection of indole



United States Patent 3,378,346 DIAGNOSTIC PREPARATION FOR THE DETECTION OF INDOLE Glenville R. Watson, East Orange, and Benjamin S.

Schwartz, Livingston, N.J., assignors to Warner-Lambert Pharmaceutical Company, Morris Plains, NJ., a corporation of Delaware No Drawing. Filed Feb. 5, 1965, Ser. No. 430,763 3 Claims. (Cl. 23-230) This invention relates to a composition for the detection of indole and relates more particularly to an improved composition for the detection of indole produced during the growth of certain micro-organisms.

The detection of the production of indole by microorganisms growing in a nutrient medium containing tryptophane is a well-known diagnostic tool for the taxonomic classification of these micro-organisms. The test for indole formation is especially useful in differentiating the large number of species included in the Enterobacteriaceae. For example, Escherichia coli produces indole and may thus be distinguished from Aerobacter aerogenes or Salmonella typhi which does not. Such differentiation is important for epidemiologic reasons since it affords early recognition of a Salmonella epidemic and because of the great differences in antibiotic sensitivity of the various species of this group. A rapid and sensitive method would be of great value in making this differentiation. However, most tests for the formation of indole are based on the use of subcultures grown for many hours in a special medium such as 1% tryptone and require the subsequent addition of an indole detecting reagent.

One of the preferred methods for the detection of indole is the Kovacs test. This test comprises growing a pure culture of the unknown organism in a special medium containing peptone and tryptophane for about 48 hours. At the end of the incubation period, the culture is extracted with amyl-alcohol in the presence of hydrochloric acid. The presence of indole is indicated by the development of a red color when a 5% p-dimethylaminobenzaldehyde solution is added to the amyl-alcohol extract.

Another modification is the Vracko method [American Journal of Clinical Pathology, vol. 39, No. 4, p. 429 (19 63)]. In this test, a culture grown in a nutrient medium for about 48 hours is allowed to come in contact with a strip of a suitable bibulous material containing p-dimethylaminobenzaldehyde and hydrochloric acid. While representing an improvement over the Kovacs test, this test is still far from satisfactory. Experimental evidence has indicated that due to the volatile nature of hydrochloric acid such a test strip loses its sensitivity after storage over a short period of time.

From the foregoing discussion, it is obvious that there are many disadvantages inherent in these methods. Thus, the Kovacs test requires at least 48 hours of incubation in a specialized medium. In the Vracko test the composition is not sufficiently stable over a prolonged period of time to enable the detection of indole to be achieved accurately and reliably.

Accordingly, a primary object of this invention is to provide a simple and stable composition for the rapid detection of indole as produced by growing micro-orgal'llSIIlS.

A further object of this invention is to provide a simple and rapid process for separating and differentiating indole producing organisms from non-indole producing organisms.

Another object of this invention is to provide a single, stable reagent composition in dry form impregnated on to a carrier strip which is suitable for the rapid and accurate detection of the presence of indole.

3,378,346 Patented Apr. 16, 1968 Other objects and advantages of this invention will become apparent from the following detailed description.

We have now found that the aforementioned objects are fulfilled by providing a strip of a bibulous material which is impregnated over at least a portion of its area with a reagent system comprising p-dimethylaminobenzaldehyde and oxalic acid. The oxalic acid acts as a stabilizing agent and in the system provided prevents the decomposition of the p dimethylaminobenzaldehyde. Since the oxalic acid is non-volatile, it further stabilizes the composition which results in an enhanced and greatly extended shelf life.

The reagent system is prepared for application to a carrier material by first dissolving oxalic acid in a lower aliphatic alcohol such as methanol or ethanol to form a 10 to 40% by weight solution and to this solution is added sufficient p-dimethylaminobenzaldehyde to form a 1 to 10% by Weight solution. The resulting alcohol solution of oxalic acid and p-dirnethylaminobenzaldehyde is then allowed to saturate all or only a selected portion of a suitable carrier material. After the alcohol solvent has evaporated, it may be desirable in some instances to apply a barrier zone to prevent capillary migration of the reagent beyond a certain point or outside of a predetermined zone, and this may be done by applying a hydrophobic material such as a lacquer to the bibulous carrier. The increase in dry weight of the carrier material prior to any barrier application is from about 10 to 30% by weight due to the reagents thus applied.

Suitable bibulous material for use as carriers are those materials which are able to draw a liquid upward by means of capillary action. Materials such as filter paper, felt, porous ceramic strips, woven or matted glass fibers or the like are suitable. The absorbency of the carrier materials may be enhanced by coating them with a solution of a polymeric substance such as hydroxy methyl cellulose.

In use, the area of the bibulous material impregnated with the reagent system is allowed to come in contact with a suspension of a pure culture of an unknown microorganism contained in a test tube, for example, and the bibulous material absorbs a portion of the suspension. A positive test is indicated by the formation of a pink to red color which appears within 5 to 10 seconds.

The advantages and convenience of the above-described invention are quite outstanding since the test for indole production can be carried out in a very much shorter time than was previously considered necessary. Moreover, the use of a special medium and testing procedure is obviated by the use of the novel dry test strip described. In addition, because of the enhanced stability of the impregnating composition, its sensitivity for detecting indole remains unchanged and even after prolonged storage, no loss of sensitivity has been observed.

The following examples are included in order to further illustrate the invention.

EXAMPLE 1 Preparation of the reagent system 66.0 grams of oxalic acid are dissolved in sufiicient ethyl alcohol to give a final volume of 260 ml. The resulting solution is heated to about 45 0., about 2.7 grams of p-dimethylarninobenzaldehyde are added and the solution is then allowed to cool.

EXAMPLE 2 Preparation of the test strip Suitable bibulous material such as Whatman No. 3 filter paper is saturated and thoroughly impregnated with the reagent system prepared in accordance with Example 1. The wetted filter paper is allowed to air dry at room temperature and the increase in dry weight is found to be about 20%. To facilitate packaging into bottles, it is cut into small strips having a length of about 4 inches and a width of about inch.

EXAMPLE 3 Use of the reagent strips One to two loopfuls of the unknown bacteria which has previously been grown on a nutrient agar slant or a nutrient broth containing tryptophane are placed on the area of the strip which has been impregnated with the reagent system. A pink to red color develops within five to ten seconds indicating the presence of indole.

It is understood that the foregoing detailed description is given merely by way of illustration and that many variations may be made therein without departing from the spirit of our invention.

Having described our invention, what we desire to secure by Letters Patent is:

1. A diagnostic preparation for the rapid and positive detection of indole which comprises a strip of a bibulous material impregnated with p-dimethylaminobenzaldehyde and oxalic acid.

2. A diagnostic preparation for the rapid and positive detection of indole which comprises a dry strip of a bibulous material previously impregnated with a 10 to 40% by weight solution of oxalic acid and a 1 to 10% by weight solution of p-dimethylaminobenzaldehyde in an inert volatile solvent followed by drying.

3. Process for the detection of indole produced by microorganisms which comprises allowing the test strip as defined in claim 2 to come in contact with an unknown organism and observing the color formed.

References Cited Knowlton et 211., Use of a Modified Ehrlichs Reagent for Measurement of Indolic Compounds, Analytical Chemistry, vol. 32, No. 6, May 1960, pp. 666-668.

MORRIS O. WOLK, Primary Examiner.

R. E. SERWIN, Assistant Examiner. 

2. A DIAGNOSTIC PREPARATION FOR THE RAPID AND POSITIVE DETECTION OF INDOLE WHICH COMPRISES A DRY STRIP OF A BIBULOUS MATERIAL PREVIIOUSLY IMPREGNATED WITH A 10 TO 40% BY WEIGHT SOLUTION OF OXALIC ACID AND A 1 TO 10% BY WEIGHT SOLUTION OF P-DIMETHYLAMINOBENZALDEHYDE IN AN INERT VOLATILE SOLVENT FOLLOWED BY DRYING.
 3. PROCESS FOR THE DETECTION OF INDOLE PRODUCED BY MICROORGANISMS WHICH COMPRISES ALLOWING THE TEST STRIP AS DEFINED IN CLAIM 2 TO COME IN CONTACT WITH AN UNKNOWN ORGANISM AND OBSERVING THE COLOR FORMED. 